The present invention relates to chimeric regulatory regions useful for controlling the expression of genes in plants. These chimeric regulatory regions can be derived from the opine synthase genes of the plant pathogen Agrobacterium tumefaciens.
Agrobacterium tumefaciens is a Gram-negative soil bacterium that infects most dicotyledonous and some monocotyledonous plants. An infection by Agrobacterium tumefaciens often results in the formation of crown gall tumors on the infected plant.
During the A. tumefaciens infection process, a defined DNA segment ("T-DNA") of the large tumor-inducing ("Ti") plasmid is transferred to a susceptible plant cell and integrated into the plant nuclear genome, whereby the T-DNA genes are expressed. Some T-DNA genes encode enzymes involved in the synthesis of hormones that are active in plants. These hormones can cause tumors in infected plants. Other T-DNA genes direct the synthesis and secretion of unique amino acid and sugar derivatives, termed opines. Agrobacterium tumefaciens can utilize these opines as a carbon and sometimes a nitrogen source. See Gelvin, Plant Physiol. 92: 281-85 (1990); Gelvin, TRANSGENIC PLANTS (Academic Press 1993); Ream, Ann. Rev. Phytopathol. 27: 583-618 (1989); Zambryski, Ann. Rev. Plant Physiol. Plant Mol. Biol. 43: 465-90 (1992).
T-DNA genes contain regions that are functional in plant environments and possess similarities to plant regulatory regions. For example, most plant promoters contain cis-acting elements such as upstream activating sequences ("UAS") (often called "enhancers") that, by binding trans-acting factors, define or influence the promoter strength and tissue-specific expression pattern. Atchison, Annu. Rev. Cell Biol. 4: 127-53 (1988). The overall strength of a given promoter, as well as its pattern of expression, can be influenced by the combination and spatial orientation of cis-acting elements and the presence of the nuclear factors that interact with these elements. Dynan, Cell 58: 1-4 (1989). Although initially resident on a prokaryotic plasmid, T-DNA genes possess all of the sequence elements (promoters and UAS) required for transcription in plants. For instance, T-DNA genes contain TATA boxes that set the site of transcription initiation, and often contain upstream elements, located more than 100 bp from the transcription initiation site, that modulate the levels of transcription. See Gelvin, TRANSGENIC PLANTS (Academic Press 1993).
Two T-DNA genes that possess upstream activating sequences are the octopine synthase (ocs) and mannopine synthase (mas) genes. The ocs gene encodes a product that condenses arginine and pyruvate to form octopine. Hack and Kemp, Plant Physiol. 65: 949-55 (1980). A 16-base pair palindrome located upstream of the ocs gene is capable of activating a heterologous maize adh1 promoter in a transient expression system. Ellis et al., EMBO J. 6: 11-16 (1987); Ellis et al., EMBO J. 6: 3203-08 (1987). This palindrome is also essential for ocs promoter activity in stably transformed tobacco calli. Leisner and Gelvin, Proc. Nat'l Acad. Sci. USA 85: 2553-57 (1988); Leisner and Gelvin, Plant Cell 1: 925-36 (1989).
The mas 1' and 2' genes share a dual bidirectional promoter and a 479 bp intergenic region. These genes encode enzymes for a two-step pathway for the synthesis of mannopine. Ellis et al., Mol. Gen. Genet. 195: 466-73 (1984); Komro et al., Plant Mol. Biol. 4: 253-63 (1985). The transcription of the mas genes is divergent, and the intergenic region contains all the cis-acting elements necessary for the transcription of both genes. DiRita and Gelvin, Mol. Gen. Genet. 207: 233-41 (1987); Fox et al. Plant Mol. Biol. 20: 219-33 (1992); Leung et al., Mol. Gen. Genet. 230: 463-74 (1991); Guevara-Garcia et al., Plant J. 4: 495-505 (1993).
The ocs and mas gene promoters have been used to direct the expression of linked genes in transgenic plants. However, the application of these promoters has been restricted by weak expression levels in certain tissues of transgenic plants. DiRita and Gelvin, supra; Harpster et al., Mol. Gen. Genet. 212: 182-90 (1988); Sanger et al., Plant Mol. Biol. 14: 433-43 (1990). For example, the ocs promoter directs a distinct cell-specific pattern of expression in transgenic tobacco. Kononowicz et al., Plant Cell 4: 17-27 (1992). The mas gene exhibits weak expression in leaves and stems, but has stronger expression in roots and exhibits a degree of wound and auxin inducibility. Langridge et al., Proc. Nat'l Acad. Sci 86: 7890-94 (1989); Teeri et al., EMBO J., 8: 343-50 (1989); Saito et al., Planta 184: 40-46 (1991); Guevara-Garcia et al., loc. cit.
Because promoters and other regulatory regions exhibit varying strengths and tissue specificities, certain recombinant regulatory regions have been developed. For example, enhancer elements that specifically bind certain trans-acting factors can modulate the transcriptional activity and the cell-specific expression pattern. Bienz and Pelham, Cell 45: 753-60 (1986).
The use of certain constitutive promoters, such as cauliflower mosiac virus (CaMV) 35S constructs, is also known. The CaMV 35S promoter has activators with multiple domains that can function to activate the 35S promoter in a developmentally and tissue-specific manner. See Benfey et al., EMBO J. 8: 2195-2202 (1989); Benfey, et al. EMBO J. 9: 1677-1684 (1990); Benfey et al., EMBO J. 9: 1685-96.
Koziel et al., Bio/Technology 11: 194-199 (1993) generally relates to promoters used in a promoter stacking construction in an effort to obtain tissue-specific promotion of a heterologous gene. Koziel shows construction of a gene expression system comprising a truncated cryIA(b) gene (the gene fragment used encodes the first 648 amino acids of an 1155 amino acid insecticidal protein from Bacillus thuringiensis) connected to either a CaMV 35S promoter or to a combination of two tissue-specific promoters derived from corn (phosphoenol-pyruvate carboxylase ("PEPC") promoter and a pollen specific promoter). Koziel reports high levels of expression from either promoter configuration. Koziel et al. also used (1) the PEPC promoter known to cause green tissue-specific expression and (2) a maize pollen-specific promoter. The expression of the insecticidal protein ranged from 1500-4000 ng/mg protein observed which appears to be a quite high level of expression. Additionally, use of the PEPC/pollen-specific promoters resulted in tissue-specific expression.
Others have attempted recombinant expression by other techniques. Bevan et al., PCT/GB92/00566 (1992), generally relates to a non-stacking application of a single promoter, which is different from the promoter of Koziel et al., to obtain tissue specific expression of a heterologous gene. This application apparently relates to the use of the bean phenylalanine ammonia lyase ("PAL") promoter to provide tissue-specific expression. A hybrid gene was constructed which fused the "putative transcriptional regulatory regions" of a genomic clone of PAL (that is, a clone which contains in addition to the coding sequences, intervening sequences which are part of the PAL gene sequence) and an open reading frame ("ORF") comprising 68 amino acids of the amino-terminal PAL protein with the entire ORF of .beta.-glucuronidase ("GUS") followed by a polyadenylation and transcriptional termination region of nopaline synthase (the later two elements are typically fused in most eukaryotic plant expression vectors to ensure efficient expression). While this initial construct did exhibit strong activity in some plants, the researchers thereafter created deletions in the PAL promoter regions of the hybrid gene. The minimum PAL promoter necessary for full expression was found to be 253 base pairs from the transcriptional start site of PAL. By varying the deletion patterns chiefly within this minimal region, the researchers found that tissue-specific expression could be modified to a degree.
U.S. Pat. No. 5,034,322 generally relates to use of nopaline synthase promoters with a ribulose-1.5-bis-phosphate carboxylase small subunit gene.
It has also been reported that a chimeric promoter called "Mac," which incorporates the mas region from +65 to -301 and the 35S enhancer region from -90 to -941, shows GUS activity at a level several times that of a double CaMV 35S promoter in transgenic tobacco plants Comai et al., Plant Mol. Biol. 15: 373-81 (1990).
The above-described constructs have exhibited several limitations in terms of expression efficiency and controllability. For example, prior approaches have failed to provide strong expression in a constitutive-like manner in circumstances where such expression is desired.